Summary
The unfolded protein response (UPR) allows cells to cope with endoplasmic reticulum (ER) stress induced by accumulation of misfolded proteins in the ER. Due to its sensitivity to ...Agrobacterium tumefaciens, the model plant Nicotiana benthamiana is widely employed for transient expression of recombinant proteins of biopharmaceutical interest, including antibodies and virus surface proteins used for vaccine production. As such, study of the plant UPR is of practical significance, since enforced expression of complex secreted proteins often results in ER stress. After 6 days of expression, we recently reported that influenza haemagglutinin H5 induces accumulation of UPR proteins. Since up‐regulation of corresponding UPR genes was not detected at this time, accumulation of UPR proteins was hypothesized to be independent of transcriptional induction, or associated with early but transient UPR gene up‐regulation. Using time course sampling, we here show that H5 expression does result in early and transient activation of the UPR, as inferred from unconventional splicing of NbbZIP60 transcripts and induction of UPR genes with varied functions. Transient nature of H5‐induced UPR suggests that this response was sufficient to cope with ER stress provoked by expression of the secreted protein, as opposed to an antibody that triggered stronger and more sustained UPR activation. As up‐regulation of defence genes responding to H5 expression was detected after the peak of UPR activation and correlated with high increase in H5 protein accumulation, we hypothesize that these immune responses, rather than the UPR, were responsible for onset of the necrotic symptoms on H5‐expressing leaves.
Agrobacterium‐mediated expression of influenza haemagglutinins allows for the production of plant‐made vaccines via accumulation of virus‐like particles. In plant cells biofactories, this molecular farming process results in early and transient activation of the unfolded protein response, which likely favors recombinant protein expression.
Summary
The production of influenza vaccines in plants is achieved through transient expression of viral hemagglutinins (HAs), a process mediated by the bacterial vector Agrobacterium tumefaciens. HA ...proteins are then produced and matured through the secretory pathway of plant cells, before being trafficked to the plasma membrane where they induce formation of virus‐like particles (VLPs). Production of VLPs unavoidably impacts plant cells, as do viral suppressors of RNA silencing (VSRs) that are co‐expressed to increase recombinant protein yields. However, little information is available on host molecular responses to foreign protein expression. This work provides a comprehensive overview of molecular changes occurring in Nicotiana benthamiana leaf cells transiently expressing the VSR P19, or co‐expressing P19 and an influenza HA. Our data identifies general responses to Agrobacterium‐mediated expression of foreign proteins, including shutdown of chloroplast gene expression, activation of oxidative stress responses and reinforcement of the plant cell wall through lignification. Our results also indicate that P19 expression promotes salicylic acid (SA) signalling, a process dampened by co‐expression of the HA protein. While reducing P19 level, HA expression also induces specific signatures, with effects on lipid metabolism, lipid distribution within membranes and oxylipin‐related signalling. When producing VLPs, dampening of P19 responses thus likely results from lower expression of the VSR, crosstalk between SA and oxylipin pathways, or a combination of both outcomes. Consistent with the upregulation of oxidative stress responses, we finally show that reduction of oxidative stress damage through exogenous application of ascorbic acid improves plant biomass quality during production of VLPs.
Biopharmaceutical glycoproteins produced in plants carry N‐glycans with plant‐specific residues core α(1,3)‐fucose and β(1,2)‐xylose, which can significantly impact the activity, stability and ...immunogenicity of biopharmaceuticals. In this study, we have employed sequence‐specific transcription activator‐like effector nucleases (TALENs) to knock out two α(1,3)‐fucosyltransferase (FucT) and the two β(1,2)‐xylosyltransferase (XylT) genes within Nicotiana benthamiana to generate plants with improved capacity to produce glycoproteins devoid of plant‐specific residues. Among plants regenerated from N. benthamiana protoplasts transformed with TALENs targeting either the FucT or XylT genes, 50% (80 of 160) and 73% (94 of 129) had mutations in at least one FucT or XylT allele, respectively. Among plants regenerated from protoplasts transformed with both TALEN pairs, 17% (18 of 105) had mutations in all four gene targets, and 3% (3 of 105) plants had mutations in all eight alleles comprising both gene families; these mutations were transmitted to the next generation. Endogenous proteins expressed in the complete knockout line had N‐glycans that lacked β(1,2)‐xylose and had a significant reduction in core α(1,3)‐fucose levels (40% of wild type). A similar phenotype was observed in the N‐glycans of a recombinant rituximab antibody transiently expressed in the homozygous mutant plants. More importantly, the most desirable glycoform, one lacking both core α(1,3)‐fucose and β(1,2)‐xylose residues, increased in the antibody from 2% when produced in the wild‐type line to 55% in the mutant line. These results demonstrate the power of TALENs for multiplexed gene editing. Furthermore, the mutant N. benthamiana lines provide a valuable platform for producing highly potent biopharmaceutical products.
Objectives:
Rechallenge/continuation of clozapine in association with colony-stimulating factors (CSFs) following neutropenia/agranulocytosis has been reported, but many questions remain unanswered ...about efficacy and safety. This systematic review aims to assess the efficacy and safety of rechallenging/continuing clozapine in patients following neutropenia/agranulocytosis using CSFs.
Methods:
MEDLINE, Embase, PsycInfo, and Web of Science databases were searched from inception date to July 31, 2022. Articles screening and data extraction were realized independently by two reviewers, according to Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) 2020 systematic review guidance. Included articles had to report on at least one case where clozapine was rechallenged/continued using CSFs despite previous neutropenia/agranulocytosis.
Results:
Eight hundred forty articles were retrieved; 34 articles met the inclusion criteria, totaling 59 individual cases. Clozapine was successfully rechallenged/continued in 76% of patients for an average follow-up period of 1.9 years. There was a trend toward better efficacy reported in case reports/series, compared with consecutive case series (overall success rates of 84% and 60%, respectively, p-value = 0.065). Two administration strategies were identified, “as-needed” and prophylactic, both yielding similar success rates (81% and 80%, respectively). Only mild and transient adverse events were documented.
Conclusions:
Although limited by the relatively small number of published cases, factors such as time of onset to first neutropenia and severity of the episode did not seem to impact the outcome of a subsequent clozapine rechallenge using CSFs. While the efficacy of this strategy remains to be further adequately evaluated in more rigorous study designs, its long-term innocuity warrants considering its use more proactively in the management of clozapine hematological adverse events as to maintain this treatment for as many individuals as possible.
Introduction
Problem gambling (PBG) is more common in people with mental health disorders, including substance use, bipolar, and personality disorders, than in the general population. Although ...individuals with psychotic disorders might be expected to be more vulnerable to PBG, fewer studies have focused on this comorbidity. The aim of this review was to estimate the prevalence of PBG in people with psychotic disorders.
Methods
Medline (Ovid), EMBASE, PsycINFO (Ovid), CINAHL, CENTRAL, Web of science, and ProQuest were searched on November 1, 2023, without language restrictions. Observational and experimental studies including individuals with psychotic disorders and reporting the prevalence of PBG were included. Risk of bias was assessed using the Joanna Briggs Institute critical appraisal for systematic reviews of prevalence data. The pooled prevalence of PBG was calculated using a fixed effects generalized linear mixed model and presented through forest plots.
Results
Of 1271 records screened, 12 studies (n = 3443) were included. The overall prevalence of PBG was 8.7% (95% CI = 7.8%–9.7%, I2 = 69%). A lower prevalence was found in studies with a low risk of bias (5.6%; 95% CI = 4.4%–7.0%) compared with studies with a moderate risk of bias (10.4%; 95% CI = 9.2%–11.7%). Different methods used to assess PBG also contributed to the heterogeneity found.
Conclusion
This meta‐analysis found substantial heterogeneity, partly due to the risk of bias of the included studies and a lack of uniformity in PBG assessment. Although more research is needed to identify those at increased risk for PBG, its relatively high prevalence warrants routine screening for gambling in clinical practice.
Arginine-rich cell penetrating peptides are short cationic peptides able to cross biological membranes despite their peptidic character. In order to optimize their penetration properties and further ...elucidate their mechanisms of cellular entry, these peptides have been intensively studied for the last two decades. Although several parameters are simultaneously involved in the internalization mechanism, recent studies suggest that structural modifications influence cellular internalization. Particularly, backbone rigidification, including macrocyclization, was found to enhance proteolytic stability and cellular uptake. In the present work, we describe the synthesis of macrocyclic arginine-rich cell penetrating peptides and study their cellular uptake properties using a combination of flow cytometry and confocal microscopy. By varying ring size, site of cyclization, and stereochemistry of the arginine residues, we studied their structure–uptake relationship and showed that the mode and site of cyclization as well as the stereochemistry influence cellular uptake. This study led to the identification of a hepta-arginine macrocycle as efficient as its linear nona-arginine congener to enter cells.
Experimental and clinical evidence supports the role of inflammation in atherosclerosis and its complications. Colchicine is an orally administered, potent antiinflammatory medication that is ...indicated for the treatment of gout and pericarditis.
We performed a randomized, double-blind trial involving patients recruited within 30 days after a myocardial infarction. The patients were randomly assigned to receive either low-dose colchicine (0.5 mg once daily) or placebo. The primary efficacy end point was a composite of death from cardiovascular causes, resuscitated cardiac arrest, myocardial infarction, stroke, or urgent hospitalization for angina leading to coronary revascularization. The components of the primary end point and safety were also assessed.
A total of 4745 patients were enrolled; 2366 patients were assigned to the colchicine group, and 2379 to the placebo group. Patients were followed for a median of 22.6 months. The primary end point occurred in 5.5% of the patients in the colchicine group, as compared with 7.1% of those in the placebo group (hazard ratio, 0.77; 95% confidence interval CI, 0.61 to 0.96; P = 0.02). The hazard ratios were 0.84 (95% CI, 0.46 to 1.52) for death from cardiovascular causes, 0.83 (95% CI, 0.25 to 2.73) for resuscitated cardiac arrest, 0.91 (95% CI, 0.68 to 1.21) for myocardial infarction, 0.26 (95% CI, 0.10 to 0.70) for stroke, and 0.50 (95% CI, 0.31 to 0.81) for urgent hospitalization for angina leading to coronary revascularization. Diarrhea was reported in 9.7% of the patients in the colchicine group and in 8.9% of those in the placebo group (P = 0.35). Pneumonia was reported as a serious adverse event in 0.9% of the patients in the colchicine group and in 0.4% of those in the placebo group (P = 0.03).
Among patients with a recent myocardial infarction, colchicine at a dose of 0.5 mg daily led to a significantly lower risk of ischemic cardiovascular events than placebo. (Funded by the Government of Quebec and others; COLCOT ClinicalTrials.gov number, NCT02551094.).
Traditional approaches for finding well-performing parameterizations of complex imaging systems, such as super-resolution microscopes rely on an extensive exploration phase over the illumination and ...acquisition settings, prior to the imaging task. This strategy suffers from several issues: it requires a large amount of parameter configurations to be evaluated, it leads to discrepancies between well-performing parameters in the exploration phase and imaging task, and it results in a waste of time and resources given that optimization and final imaging tasks are conducted separately. Here we show that a fully automated, machine learning-based system can conduct imaging parameter optimization toward a trade-off between several objectives, simultaneously to the imaging task. Its potential is highlighted on various imaging tasks, such as live-cell and multicolor imaging and multimodal optimization. This online optimization routine can be integrated to various imaging systems to increase accessibility, optimize performance and improve overall imaging quality.
Control over glycosylation is an important quality parameter in recombinant protein production. Here, we demonstrate the generation of a marker‐free genome edited Nicotiana benthamiana ...N‐glycosylation mutant (NbXF‐KO) carrying inactivated β1,2‐xylosyltransferase and α1,3‐fucosyltransferase genes. The knockout of seven genes and their stable inheritance was confirmed by DNA sequencing. Mass spectrometric analyses showed the synthesis of N‐glycans devoid of plant‐specific β1,2‐xylose and core α 1,3‐fucose on endogenous proteins and a series of recombinantly expressed glycoproteins with different complexities. Further transient glycan engineering towards more diverse human‐type N‐glycans resulted in the production of recombinant proteins decorated with β1,4‐galactosylated and α2,6‐sialylated structures, respectively. Notably, a monoclonal antibody expressed in the NbXF‐KO displayed glycosylation‐dependent activities. Collectively, the engineered plants grow normally and are well suited for upscaling, thereby meeting industrial and regulatory requirements for the production of high‐quality therapeutic proteins.
Graphical and Lay Summary
Engineering glycosylation pathway in plants increases quality of recombinantly expressed proteins. A Nicotiana benthamiana line that lacks active xylosyltransferase genes was used to knock out core fucosyltransferase genes by multi‐target CRISPR/Cas9‐based gene editing. The engineered line synthesizes human‐type GnGn structures well suited for industrial‐scale production of proteins with homogeneous N‐glycans and serves as template for further glycan diversifications.
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